Project Summary Genome-wide functional genetics has been recently made possible by pooled library-based CRISPR/Cas9 gene editing technology. By randomly knocking out thousands of genes, large-scale screens can be conducted for functional genetics, drug sensitivity and other phenotypes. Currently however, this forward genetic approach is largely relegated to live/dead screens in mammalian cells. Improving the sophistication of ?phenotypes-of-interest? in these screens beyond live/dead would significantly broaden the utility of guide RNA (gRNA) library screens. Here, Cell Microsystems proposes the use of its proprietary CellRaft? Technology to automate imaging-based CRISPR/Cas9 screening. The CellRaft Technology comprises the CytoSort? Array, which contains 10?s of thousands of microwells where cells randomly distribute. Once seeded, cells can be can be transfected with Cas9 and gRNA libraries. After undergoing CRISPR/Cas9-based gene editing, cells can be screened using the AIR? System, an instrument designed to image, sort and isolate cells from the CytoSort Array. Cells exhibiting the phenotype-of-interest can then be collected individually for either molecular analysis or clonal propagation. To pursue these goals, Cell Microsystems will collaborate with William Buchser, PhD of Washington University (St. Louis). His core facility has extensive expertise in genome-wide screening and has identified the CellRaft Technology as a solution for both accelerating gRNA-based screening and employing sophisticated phenotype targets. For Phase I feasibility testing (Aim 1) of the CellRaft Technology for gRNA panel screening, we will employ a CRISPR screen against cells expressing various fluorescent proteins. Cells will be transfected with Cas9 and a pool of gRNAs against each transgenic fluorescent protein and control gRNAs. This method allows a quantitative assessment of both CRISPR/Cas9 efficiency and imaging-based sorting of cells prior to isolation using the CellRaft Technology. In a parallel program, Cell Microsystems has already demonstrated successful CRISPR/Cas9 genome editing using the CellRaft Technology, which integrates the entire genome editing workflow including transfection, imaging-based screening of cells, harvesting? and clonal colony growth. These capabilities are all essential to streamlining gRNA screening methods and enable investigators to sort cells for relatively subtle phenotypes. The imaging capabilities of the AIR? System are sufficient for both cellular morphology analysis, sub-cellular localization of fluorescent signals, and cell-cell interactions?all key criteria for screening sophisticated cellular phenotypes. The proposed program also includes the development of appropriate image analysis and cell sorting software to monitor the growth of clonal colonies. Tailoring several features of the CellRaft Technology, already a staple for single cell isolation in many labs, will address the currently unmet needs of large-scale pooled gRNA CRISPR screening workflows.